DNA methylation of miR-138 regulates cell proliferation and EMT in cervical cancer by targeting EZH2

Background Emerging evidence has identified miR-138 as a tumor suppressor that can suppress the proliferation of various cancers. Meanwhile, the cause of abnormal miR-138 expression in cervical cancer remains uncertain. This study clarified the mechanism by which miR-138 regulates proliferation, invasion, metastasis, and EMT in cervical cancer cells. Results miR-138 expression in human cervical cancer and adjacent normal tissue was measured using qPCR. SiHa and C33A cells were used to determine the function of miR-138 via miR-138 mimic or inhibitor transfection, followed by wound healing, Cell Counting Kit-8, flow cytometry, and Transwell assays. Epithelial and mesenchymal marker expression was analyzed using Western blotting. DNA methylation in the miR-138 promoter was examined using bisulfite sequencing PCR. The downstream target genes of miR-138 were identified via bioinformatics analysis and luciferase reporter assays. A tumor xenograft model was employed to validate DNA methylation-induced miR-138 downregulation and tumor growth inhibition in cervical cancer in vivo. miR-138 levels were significantly lower in cervical cancer tissues than in adjacent control tissues. Furthermore, lower miR-138 expression and higher CpG methylation in the miR-138 promoter were identified in lymph node-positive metastatic cervical cancer tumors versus that in non-metastatic tumor tissues. Upon miR-138 overexpression, cell proliferation, metastasis, invasion, and EMT were suppressed. miR-138 agomir transfection and demethylating drug treatment significantly inhibited cervical tumor growth and EMT in tumor xenograft models. DNA methylation inhibited miR-138 transcription, and enhancer of zeste homolog 2 (EZH2) downregulation mediated the tumor suppressor function of miR-138 in cervical cancer. Conclusion We demonstrated that miR-138 suppresses tumor progression by targeting EZH2 in cervical cancer and uncovered the role of DNA methylation in the miR-138 promoter in its downregulation. These findings demonstrated the potential of miR-138 to predict disease metastasis and/or function as a therapeutic target in cervical cancer. Supplementary Information The online version contains supplementary material available at 10.1186/s12885-022-09477-5.


Introduction
Cervical cancer has become the fourth most common malignant tumor in females worldwide, and it carries a high mortality rate and poor prognosis. Although immunization against human papillomavirus (HPV) and the improvement of cervical cancer screening have led to significant decreases of the incidence of cervical cancer, the prognosis of patients remains poor because of lymph node metastasis and pelvic invasion [1][2][3][4]. Thus, identification of the key factors that regulate epithelial-mesenchymal transition (EMT) and metastasis in cervical cancer is urgently required.
MicroRNAs (miRNAs) are short RNAs (20-22 nucleotides) that do not encode proteins [5]. The first miRNA was found from caenorhabditis elegans in 1993 and the mammalian miRNA was discovered in 2000 [6,7]. The genes encoding miRNA in the nucleus are transcribed into primary miRNA (pri-miRNA). Pri-miRNA was cleaved into stem-loop precursors(pre-miRNA) of approximately 70 nucleotides under the action of Drosha RNase. Pre-miRNA are exported from the nucleus to the cytoplasm in Ran-GTP-dependent Exportin 5. Under the action of Dicer enzyme (double-stranded RNA-specific RNA endonuclease), pre-miRNA is cleaved into 20-22 double-stranded miRNA [8,9]. Mature miRNA bind to their complementary sequences to form a double helix structure. The double helix is then unwound, and one of them binds to the RNA-induced silencing complex (RISC) to form asymmetric RISC. The complex binds to the target mRNA. In most cases, the single stranded miRNA in the complex is not fully complementary to the 3′-untranslated region (UTR) of the target mRNA, thus blocking the translation process of the gene. In addition, miRNAs have been demonstrated to play important roles in proliferation, apoptosis, invasion, differentiation, and other processes [10]. Increasing numbers of studies have described miRNA dysregulation in many human cancers and the involvement of miRNAs in the regulation of cancer occurrence, development, and metastasis [11].
Emerging evidence has identified miR-138 as a tumor suppressor capable of inhibiting cell proliferation in various cancers including glioma, ovarian cancer, liver cancer, lung cancer, kidney cancer, and prostate cancer [12][13][14][15][16][17]. We previously found 18 differentially expressed miRNAs in HPV16-positive cervical cancer tissues, including miR-138, which was downregulated in cancer [18]. However, few reports have described the regulatory roles of miR-138 in metastasis and EMT in cervical cancer.
EMT describes a biological process whereby epithelial cells lose their polarization and adherence and acquire mesenchymal-like migratory and invasive characteristics [19]. The cellular epithelial status is mainly dependent on the calcium-dependent E-cadherin transmembrane adhesion molecule, which is responsible for sustaining connections between adjacent cells. During EMT, cells undergo a series of changes in gene expression, functionality, and morphology that are associated with decreased E-cadherin expression and increased N-cadherin expression [20,21]. EMT is considered the pre-step of cancer cell metastasis. Meanwhile, several studies have identified enhancer of zeste homolog 2 (EZH2) as a positive upstream regulator of the EMT program. EZH2 can combine with the CDH1 (encoding E-cadherin) promoter to decrease the expression of E-cadherin and promote the metastasis and invasion of cancer cells [22].
DNA methylation, as one of the most common epigenetic regulations, is an important regulator of gene expression, cell apoptosis, tumorigenesis, and differentiation [23,24]. Accumulated data indicate that DNA methylation can promote the downregulation of miRNAs, thereby regulating tumor development and progression [25]. The DNA methylation-mediated control of miRNA expression, such as miR-200b hypomethylation and miR-124 hypermethylation, has been reported in cervical cancer [26,27].
In this study, we assessed the expression and function of miR-138 in cervical cancer. Low miR-138 expression was associated with increased levels of CpG methylation in the miR-138 promoter in tumors from patients with lymph node positivity or lymphatic space invasion compared with the findings in non-metastatic tumor tissues. miR-138 overexpression inhibited cell proliferation, invasion, migration, and EMT in cervical cancer. miR-138 agomir and a demethylating drug significantly inhibited cervical tumor growth and EMT in xenograft models. We identified EZH2 as an miR-138 target in this cancer type. Methylation of the miR-138 promoter transcriptionally suppressed its expression. These findings suggested the potential of miR-138 to predict disease metastasis and/or serve as a therapeutic target for the treatment of cervical cancer.

Clinical samples
For this study, we utilized 15 pairs of cervical cancer and paracancerous tissues and 83 tumor tissues collected from patients diagnosed with cervical cancer who were treated at Affiliated Tumor Hospital of Xinjiang Medical University between 2012 and 2014. World Health Organization classifications were used for the histopathological diagnosis of these patients, with staging conducted using the criteria of the International Federation of Gynecology and Obstetrics. The Ethics Committee of the Affiliated Tumor Hospital of Xinjiang Medical University approved the present study. All patients have signed the written informed consent forms before surgical resection. Following collection, tissue samples were snap-frozen and stored at − 80 °C prior to use.

Cell culture and reagents
SiHa and C33A cells were obtained from Institute of Cell Research, Chinese Academy of Sciences (Shanghai, China) and grown in DMEM (SiHa) or MEM (C33A) containing 10% FBS and penicillin/streptomycin at 37 °C in a 5% CO 2 atmosphere. All cell culture reagents were procured from Gibco (USA).

Cell transfection
Negative-control miRNA (miR-NC) and an miR-138 mimic and inhibitor were acquired from Shanghai Gene Pharmaceuticals Co., Ltd. EZH2 siRNA was purchased from Shanghai USEN Biological Technology Co., Ltd. These constructs were transfected into cells using Lipofectamine ® 3000 (Thermo Fisher Scientific,USA) according to the product instruction manual. At 24 h after transfection, cells were subjected to further experiments.

qRT-PCR
An miRNeasy Mini Kit (Qiagen, Germany) and Cell lysis buffer MZ (Tiangen, Beijing, China) were used to extract RNA from samples, after which PrimeScript ™ RT reagent (Takara, Japan) and an miScript II RT Kit (Qiagen, Germany) were used to prepare cDNA based on the provided directions. Next, an miScript SYBR Green PCR Kit (Qiagen, Germany) and SYBR ® Premix Ex Taq ™ II (Tli RNaseH Plus, Takara, Japan) were used to detect miRNA and mRNA expression levels. The internal controls were U6 and β-actin (primers are listed in the Supplementary  Table). Relative expression was quantified via the − ΔCt method.

Cell proliferation assay
At 24 h after transfection, the Cell Counting Kit-8 (CCK-8) assay (Beyotime, Beijing, China) was employed to assess the proliferation of cervical cancer cells using the manufacturer's directions. In total, 2000 cells were seeded into 96-well plates with three replicates and incubated with CCK-8 reagent (10 μl) for 24, 48, 72, or 120 h at 37 °C. The relative cell viability was assessed at 450 nm using a microplate reader (Thermo Fisher Scientific, USA).

Flow cytometry
Transfected cells were plated into six-well plates (1 × 10 6 / well). Then, apoptotic cells were identified using an Annexin V-FITC Cell Apoptosis Detection Kit (Beyotime Beijing, China) and measured using a BD FACSCanto ™ II flow cytometer (BD Biosciences, USA), after which FlowJo software (v 10.4; FlowJo LLC) was used for data analysis.
For cell cycle analysis, at 24 h following transfection, cells were collected, washed thrice with PBS, and fixed overnight using 70% ethanol at 4 °C. Then, cells were resuspended in PBS and incubated with propidium iodide staining solution at 37 °C for 30 min. Cell cycle progression was evaluated via flow cytometry (BD Biosciences, USA).

Transwell assay
Cells were collected at 24 h after transfection. In total, 1 × 10 5 cells in 200 µl of serum-free medium were seeded into the upper Transwell chamber, and the lower chamber was supplemented with 800 μl of medium containing 10% FBS. Following incubation for 24 h, the upper chamber was washed thrice with PBS followed by incubation with paraformaldehyde (800 μl) for 10 min and staining using crystal violet solution (800 μl) for 15-30 min (all at room temperature). A cotton swab was utilized gently to scrape off cells attached to the upper surface of the filter membrane. Invasive cells were then enumerated via microscopy (Olympus Corporation, Japan).

Wound-healing assay
Transfected cells were seed in a six-well plate (8 × 10 5 cells/well). After overnight incubation, cells were wounded using a pipette tip and incubated in serumfree medium for 0, and 24 h prior to imaging via inverted phase contrast microscopy (Olympus Corporation, Japan).

Bisulfite Sequencing PCR (BSP)
A Genomic DNA Extraction Kit (GENERAY; GK1022, Shanghai, China) was employed to extract gDNA from all samples, after which an EpiTect Fast DNA Bisulfite Kit (QIAGEN; 59,824, Germany) was employed for bisulfite conversion according to the product manual. Then, one microliters of the bisulfite modified DNA from each sample were subjected to PCR analysis in a 30 μL volume. The reaction mixture was preheated at 95 °C for 10 min and amplified using a PCR program (i.e., 40 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 40 s; and a final extension of 5 min at 72 °C). The PCR products were then subjected to clone into the pTG19-T vector (Generay, Lot:GV6021) followed by sequencing analysis (after the cloning, 10 clones from each sample were randomly selected for DNA sequencing).

Mouse tumor xenograft model
Female BALB/c nude mice (5-6 weeks old, 15-20 g) from Shanghai Slark Experimental Animal Company were housed in specific pathogen-free conditions at Shanghai East Hospital. Animals were randomized into three groups and subcutaneously injected with C33A cells (5 × 10 6 cells/mouse, seven mice per group). After 7 days, two mice from the miR-138 agomir group and two mice from the agomir NC group were excluded from the experiment for the tumor didn't grow successfully; two mice from the decitabine (DAC) group were sacrificed for cachexia. At final test time, mice in the miR-138 agomir group were injected intratumorally with 25 μl (100 μM, physiological saline configuration) of the miR-138 agomir (Ribobio; miR10000430, Guangzhou, China) every 3 days. Animals in the agomir NC group were injected with 25 μl (100 μM, physiological saline configuration) of agomir NC (Ribobio; miR4N0000002, Guangzhou, China) every 3 days. In the DAC group, mice were injected with DAC (10 mg/kg) intraperitoneally at 11, 13, and 15 days. The tumor volume was measured every 2 weeks and calculated as follows: tumor volume [mm 3 ] = (length [mm]) × (width [mm]) 2 × 0.5. All mice were anaesthetized by inhalation of 3% isoflurane and sacrificed by breaking the neck at 29 days after implantation, at which time tumors were isolated and weighed. All experiments involving animals were approved by the Animal Care and Use Committee at Tongji University and followed by Guidelines for the ethical review of laboratory animal welfare People's Republic of China National Standard GB/T 35,892-2018.

Immunohistochemistry (IHC)
IHC was performed with specific antibodies as described previously [28].

Statistical analysis
SPSS 25.0 (SPSS, Inc.) and GraphPad Prism 8.0 (Graph-Pad Software, Inc.) were employed for all statistical testing. Data are presented as the mean ± standard deviation. Relationships between miR-138 and patient clinicopathological parameters were analyzed using oneway ANOVA. An independent-samples t-test or oneway ANOVA was used to analyze possible differences between the two groups. The methylation status was analyzed comprehensively and comparatively using Biqanalyzer. P < 0.05 was the significance threshold.

Results
Downregulation of miR-138 in metastatic cervical tumor tissues from lymph node-positive patients qPCR was performed to assess miR-138 expression in cervical cancer. miR-138 levels were lower in tumor tissues than in matched paracancerous tissues in patients with cervical squamous cell carcinoma (N = 15, Fig. 1a). miR-138 levels in cancer tissues obtained from patients with lymph node metastasis or lymph vascular space invasion were lower than those in tumors without these features (N = 83, Fig. 1b and Table 1).

EZH2 is a miR-138 target gene in cervical cancer
We previously demonstrated that miR-138 suppresses proliferative and invasive activity and induces apoptotic death in vitro [28]. In this study, using the TargetScan prediction tool, we identified EZH2 as a putative target gene of miR-138 (Fig. 2a). To validate this relationship, luciferase reporter assays were applied using Luc plasmids containing WT or Mut EZH2 3′-UTR. miR-138 overexpression markedly inhibited luciferase activity in cells transfected with WT EZH2 3′-UTR, whereas no effects on Mut EZH2 3′-UTR were noted (P < 0.05; Fig. 2b). Moreover, the miR-138 mimic or inhibitor was transfected into SiHa and C33A cells, followed by qPCR and Western blotting, revealing that EZH2 mRNA expression was reduced by the miR-138 mimic and induced by the miR-138 inhibitor (Fig. 2c). Western blotting revealed decreases of EZH2 and N-cadherin expression and increases of E-cadherin expression at the protein level in miR-138 mimic-transfected cells (Fig. 2d and Supplementary Fig. 1a). Contradictory results were detected in cells transfected with the miR-138 inhibitor ( Fig. 2d and Supplementary Fig. 1a). In addition, we found that miR-138 was negative correlation with EZH2 expression in tumor tissues ( Supplementary Fig. 1b).

EZH2 mediates the regulatory activities of miR-138 in cervical cancer
We further examined whether EZH2 suppression is critical for miR-138-induced proliferation, apoptosis, and invasion in cervical cancer cells. We determined that EZH2 silencing via siRNA significantly inhibited its expression ( Fig. 3a and Supplementary Fig. 1c). Meanwhile, we observed that cell proliferation, invasion, and metastasis were inhibited upon EZH2 suppression, in addition it promoted apoptosis. The results were similar for miR-138 mimic transfection (Fig. 3b-e). We further explored the change of protein levels, observing increased E-cadherin expression (Fig. 3a). We next conducted rescue experiments by co-transfected miR-138 inhibitor and siEZH2 in SiHa and C33A cells, further elucidating the effects of miR-138 on the regulations of EZH2. As expected, miR-138 inhibitor relieved the suppression of siEZH2 on EZH2 expression ( Fig. 3f and Supplementary Fig. 1d). Furthermore, miR-138 inhibitor markedly promoted metastatic potential, but was reversed by EZH2 knockdown (Fig. 3g).

DNA methylation regulates miR-138 expression at the transcriptional level
Our data presented thus far indicated that the regulatory function of miR-138 is mediated by EZH2 expression. However, the cause of abnormal miR-138 expression remained unclear. Previous studies reported  that methylation is closely related to miRNA expression [25]. Thus, we further assessed whether methylation also might contribute to the downregulation of miR-138 in cervical cancer. Specifically, we measured the degree of methylation of CpG sites in the miR-138 promoter in cancer tissues and adjacent tissues in patients, as well as in cell lines. The BSP results clearly confirmed the methylation of a single CpG site in the promoter region of miR-138 ( Fig. 4a and Supplementary Fig. 2). We found that the extent of CpG-9 site methylation in the miR-138 promoter was higher in cancer tissues than in paracancerous tissues (Fig. 4b).
To further verify our conjecture, we treated C33A cells with DAC, finding that miR-138 expression was increased after DAC treatment (Fig. 4c). The extent of CpG-9 methylation in the miR-138 promoter was decreased in cells treated with DAC (Fig. 4d).
Relative to the effects of miR-NC and miR-138 inhibitor treatment, cell proliferation was inhibited by DAC exposure, although the effect was weaker than that of the miR-138 mimic (Fig. 4c, e). Meanwhile, cells treated with DAC and transfected with the miR-138 mimic accumulated in the G1 phase (Fig. 4f ). In addition, Transwell and woundhealing assays illustrated that cell invasion and migration were inhibited by DAC (Fig. 4g, h). Meanwhile we found that DAC can downregulate EZH2, N-cadherin, and vimentin expression and promote E-cadherin expression at the protein level ( Fig. 4i and Supplementary Fig. 1e).

Demethylation promotes miR-138 expression and inhibits cervical tumor growth in vivo
Given the antitumor activity of miR-138 in C33A cells in vitro, we further assessed whether miR-138 overexpression would have an inhibitory action in a C33A xenograft model. We found that miR-138 agomir or DAC treatment inhibited tumor growth compared with the effects of the agomir NC. Meanwhile, miR-138 agomir treatment exerted stronger antitumor effects than DAC (Fig. 5a-c), consistent with the in vitro observations. In the 4th week, the tumor volume and tumor weight of the agomir NC were 1425 ± 203 mm 3 (N = 5) and 2.15 ± 0.38 g (N = 5), respectively. In contrast, the tumor volume and tumor weight of the miR-138 agomir group were 314 ± 69 mm 3 (N = 5) and 0.55 ± 0.19 g (N = 5), respectively; the tumor volume and tumor weight of the DAC group were 723 ± 165 mm 3 (N = 5) and 0.79 ± 0.13 g (N = 5). Data were shown as mean ± std. We next evaluated miR-138 levels in xenograft tumors. As expected, miR-138 expression in miR-138 agomir-or DAC-treated tumors was higher than that in agomir NC-treated tumors, and the miR-138 agomir more strongly upregulated miR-138 expression than DAC (Fig. 5d). Further immunohistochemical staining revealed reduced numbers of EZH2-, N-cadherin-, and Vimentin-positive cells and substantially higher numbers of E-cadherin-positive cells following treatment with the miR-138 agomir or DAC (Fig. 5e). Similarly, we observed decreased EZH2 and N-cadherin expression and increased E-cadherin expression at the protein level following treatment with the miR-138 agomir or DAC (Fig. 5f ).

Discussion
In recent decades, an increasing number of studies have confirmed the tissue-specific expression patterns of miRNAs, which are usually dysregulated in various cancers, and that miRNAs are important regulators of tumorigenesis, development, and metastasis. In cervical cancer, miRNAs have been characterized as tumor suppressors or oncogenes [29,30]. Previous studies indicated that miRNAs can inhibit proliferation, invasion, and migration by suppressing related proteins in cervical cancer [31][32][33][34].
In this study, we determined that miR-138 expression was markedly decreased in tumor tissues relative to that in normal tissues, in line with previous findings [35]. In addition, we found that decreased miR-138 expression was linked to lymphatic space invasion and lymph node metastasis. Therefore, we suspected that miR-138 might participate in the proliferation, migration, and invasion of cervical cancer cells. To test this hypothesis, functional assays were conducted to assess the roles of miR-138 in vitro. We verified that miR-138 markedly suppressed cervical cancer cell proliferation and induced cell apoptosis, consistent with previous reports [35]. Meanwhile, our results indicated that miR-138 significantly decreased cervical cancer cell invasion and metastasis, supporting prior findings of the involvement of miR-138 in tumorigenesis and development. Many studies illustrated that miR-138 can regulate various signaling pathways by inhibiting the expression of multiple proteins. Bal et al. [12] demonstrated that miR-138 targeted FOXC1 to suppress lung cancer cell proliferation and invasion. Yang et al. [30] reported similar biological effects of miR-138 in bladder cancer by targeting survivin. Meanwhile, other studies affirmed that miR-138 can affect the proliferation, apoptosis, and invasion of lung, ovarian, and gastric cancer cells by targeting the expression of PD-L1, HIF-1α, SOX4, or H2AX [17,36,37]. However, the mechanisms by which miR-138 regulates cervical cancer cell metastasis and invasion remained unclear.
It is widely known that EMT is involved in tumor invasion and metastasis [38]. In some cancer cells in primary tumors, epithelial cells lose their characteristic polarity and adherence because of EMT and attain a mesenchymal phenotype that enables invasion and metastasis, and these transformed cells exhibit molecular alterations, as confirmed by reduced E-cadherin expression and increased N-cadherin and vimentin expression [39]. Several signal pathways are involved in the regulation of EMT, including TGF-β, EZH2, Notch, Wnt/β-catenin, and hepatocyte growth factor [40][41][42][43][44][45]. Previous research revealed that EZH2 can directly induce EMT by mediating E-cadherin silencing via H3K27 trimethylation [41]. In addition, many factors can up-or downregulate EZH2. For example, EZH2 can be directly activated by the MEK-ERK-Elk-1 pathway, which induces downstream transcription regulators (c-Myc and STAT1) that bind to the EZH2 promoter. [46][47][48] Meanwhile, several miRNAs have also been revealed to suppress EZH2 expression, including miR-101, miR-144, miR-26a, miR-214, miR-98, miR-25, miR-30d, and miR-199a [49][50][51][52][53][54][55]. In the present study, we demonstrated that EZH2 was inhibited by miR-138 using biological bioinformatics analyses and luciferase reporter assays. Moreover, we conducted transfection experiments to downregulate EZH2 expression in SiHa and C33A cells and determine whether EZH2 plays regulatory roles in cervical cancer. These assays demonstrated that EZH2 silencing suppressed tumor growth, in line with prior findings [15,56]. More importantly, rescue experiments revealed EZH2 suppression partially reversed the effects of miR-138 inhibitor on the metastatic potential of SiHa and C33A cells, confirming that miR-138 targeted EZH2.
We further found that the abnormal expression of miR-138 was attributable to the hypermethylation of the promoter. DNA methylation is an important epigenetic marker in various cancers, and it mostly occurs at CpG Tumor growth curves of mice in the three groups. Tumor volumes were measured every 2 weeks starting 7 days after injection. c Tumor weights in all mice. Data are presented as the mean ± standard deviation. d Quantitative RT-PCR analysis was applied to determine the relative levels of miR-138 in all tumors described in (a). e Representative images of immunohistochemical staining of enhancer of zeste homolog 2 (EZH2), E-cadherin, N-cadherin, and vimentin in tumor samples described in (a). f Western blot analysis of EZH2, E-cadherin, N-cadherin, and vimentin expression in tumor samples described in (a). Three samples in each group were randomly selected for analysis. GAPDH served as a loading control sites in the promoter region, thereby directly mediating the expression of protein-encoding genes and indirectly regulating protein expression by targeting non-coding genes such as miRNAs [57][58][59]. Previous work revealed that many miRNAs, such as miR-126, miR-146a, miR-143, and miR-155, can be downregulated via hypermethylation of their CpG islands in cervical cancer [26,60]. In our study, cancer tissues exhibited high methylation at the CpG-9 site of the miR-138 promoter compared with that in matched adjacent tissues. In addition, miR-138 expression can be regulated by demethylating agents in vitro and vivo. Together, these findings both clarified the regulatory role of miR-138 on EZH2 expression in cervical carcinogenesis and illustrated the potential use of demethylating agents in cervical cancer treatment.

Conclusions
In summary, the current study clearly identified miR-138 as a tumor suppressor in cervical cancer, and miR-138 downregulation is attributable to DNA methylation. The functional and mechanistic studies indicated that miR-138 can suppress cervical cancer cell growth and metastasis by targeting EZH2. Our findings suggest that endogenous miR-138 upregulation induced by targeting the epigenetic machinery (such as demethylating agents) or exogenous miR-138 mimic treatment may represent potential therapeutic strategies for cervical cancer.